Effect of different levels of beef bacterial microflora on the growth and survival of Escherichia coli O157:H7 on beef carcass tissue

The influence of various levels of endogenous beef bacterial microflora on the growth and survival of Escherichia coli O157:H7 on bovine carcass surface tissue was investigated. Bacterial beef microflora inoculum was prepared by enriching and harvesting bacteria from prerigor lean bovine carcass tissue (BCT) and was inoculated onto UV-irradiated prerigor BCT at initial levels of 10(5), 10(4), 10(3), and <10(3) CFU/cm2. Additional control BCT was inoculated with sterile H2O. E. coli O157:H7 was inoculated onto all tissues at an initial level of 10(2) CFU/cm2. Following a 48-h incubation at 4 degrees C, BCT was incubated up to 14 days at 4 or 12 degrees C, either aerobically or vacuum packaged. Regardless of the microflora level, there was no substantial growth of E. coli O157:H7 on BCT during storage at 4 degrees C under either aerobic or vacuum-packaged conditions. Instead, viable cell numbers at 4 degrees C remained constant, with no reduction in numbers associated with the different beef microflora levels. E. coli O157:H7 grew on all BCT stored at 12 degrees C, regardless of microflora inoculation treatment, reaching higher populations on aerobic samples than on vacuum-packaged samples in 10 days. However, the presence of the beef microflora did appear to delay the onset of growth or slow the growth of the pathogen, and E. coli O157:H7 counts on BCT without added microflora were generally higher following 7 to 10 days of 12 degrees C storage than those counts on BCT inoculated with beef microflora. These data demonstrate the importance of temperature control during meat handling and storage to prevent the outgrowth of this pathogen and indicate that proper sanitation and processing practices that prevent and reduce contamination of carcasses with E. coli O157:H7 are essential, regardless of background microflora levels.     

Growth of Escherichia coli O157:H7 in raw ground beef stored at 10 degrees C and the influence of competitive bacterial flora,strain variation,and fat level

Pure-culture broth-based models of the growth of Escherichia coli O157:H7 have been used to estimate its behavior in ground beef, even though these models have not been adequately validated for this food product. This situation limits accurate estimates of the behavior of E. coli O157:H7 in ground beef and introduces uncertainties in risk assessments. In the present study, the growth of single and multiple strains of E. coli O157:H7 were measured in retail ground beef stored at 10 degrees C for up to 12 days, and the results were compared with estimates generated by the U.S. Department of Agriculture's Pathogen Modeling Program (PMP; version 5.1). At pH 5.9, the PMP predicted a maximum population density (MPD) of 9.13 log10 CFU/g, an exponential growth rate (EGR) of 0.052 log10 CFU/h, and a lag time of 56.3 h. Similar parameter values were observed for sterilized ground beef; however, no lag phase was observed. In contrast, the mean MPD and EGR for retail ground beef were 5.09 log10 CFU/g and 0.019 log10 CFU/h, respectively, and no lag phase was observed. Both the EGR and the MPD increased with decreasing fat levels. There was low variation in the MPD and EGR parameters for the nine E. coli O157:H7 ground beef isolates. Two isolates of competitive native flora were separately added to sterilized ground beef, and the EGR and MPD decreased as the ratio of competitive flora to E. coli O157:H7 increased. For one strain, at ratios of 1:1, 10:1, and 100:1, the EGRs were 0.033, 0.025, and 0.018 log10 CFU/h, respectively, and the MPDs were 6.14, 5.08, and 4.84 log10 CFU/g, respectively. These results demonstrate that existing broth-based models for E coli O157:H7 must be validated for food and that models should consider the effects of the food matrix, the competitive microflora, and potential pathogen strain variation.     

Impact of pH enhancement on populations of Salmonella,Listeria monocytogenes,and Escherichia coli O157:H7 in boneless lean beef trimmings

Boneless lean beef trimmings were inoculated with multiple strains of salmonellae, Listeria monocytogenes, and Escherichia coli O157:H7 at levels of ca. 6 log10 CFU/g. pH enhancement with ammonia gas was then used to increase the pH of the trimmings to ca. 9.6. The product was then frozen, chipped, and compressed into blocks. pH enhancement reduced the populations of salmonellae, L. monocytogenes, and E. coli O157:H7 by approximately 4, 3, and 1 log10 cycles, respectively. After the product had been frozen and compressed into blocks, no salmonellae or E. coli O157:H7 were detectable by enumeration or after enrichment and isolation. The final populations of L. monocytogenes were reduced by ca. 3 log10 cycles relative to the initial populations. When uninoculated pH-enhanced lean boneless trimmings were blended with inoculated ground beef to a final concentration of 15% (wt/wt), pathogen populations in the ground beef were reduced by approximately 0.2 log10 cycles.     

Distribution patterns of Escherichia coli O157:H7 in ground beef produced by a laboratory-scale grinder

This study determined the distribution patterns of Escherichia coli O157:1H7 in ground beef when a contaminated beef trim was introduced into a batch of uncontaminated beef trims prior to grinding in a small-scale laboratory grinder. A beef trim (15.3 +/- 2 g) was inoculated with a rifampicin-resistant strain of E. coli O157:H7 (E. coli O157:H7rif) and introduced into a stream of noncontaminated beef (322 +/- 33 g) prior to grinding. Seven inoculum levels (6, 5, and 4 total log CFU [high]; and 3, 2, 1, and 0 total log CFU [low]) were studied in triplicate. E. coli O157:H7rif was not detected in 3.1 to 43% of the ground beef inoculated with the high levels or in 3.4 to 96.9% of the ground beef inoculated with the low levels. For all inoculum levels studied, the five ground beef fractions (each 7.8 +/- 0.6 g) with the highest pathogen levels accounted for 59 to 100% of the total pathogens detected. For all inoculum levels, there was a linear relationship between the quantity of ground beef containing E. coli O157:H7rif and the inoculum level. The quantity of E. coli O157:H7rif in the beef remaining in the grinder was proportional to the inoculum level and was related to the location in the grinder. Different components of the grinder accumulated E. coli O157:H7rif in different quantities, with the most significant accumulation being in the nut (collar) that attaches the die to the blade. This study determined specific distribution patterns of E. coli O157:H7rif after the grinding of a contaminated beef trim along with uncontaminated trims, and the results indicate that the grinding operation should be regarded as a means of distribution of microbial contamination in risk analyses of ground beef operations.     

Effects of salt, sodium pyrophosphate, and sodium lactate on the probability of growth of Escherichia coli O157:H7 in ground beef

Ground beef products are susceptible to contamination with Escherichia coli O157:H7. The objective of this study was to examine the effect of salt, sodium pyrophosphate (SPP), and sodium lactate on the probability of growth of E. coli O157:H7 in ground beef under a temperature abuse condition. Ground beef containing 0 to 2.25% salt, 0 to 0.5% SPP, and 0 to 3% lactate was inoculated with a four-strain mixture of E. coli O157:H7, vacuum packaged, and stored at 10°C for 15 days. A total of 25 combinations of the three additives, each with 20 samples, were tested. A logistic regression was used to model the probability of growth of E. coli O157:H7 (with a 1.0-log CFU/g increase during storage) as a function of salt, SPP, and lactate. The resultant probability model indicated that lactate at higher concentrations decreased the probability of growth of E. coli O157:H7 in ground beef, and the effect was more pronounced at higher salt concentrations. At salt concentrations below 1.3%, the increase of SPP concentration marginally increased the growth probabilities of E. coli O157:H7. The model illustrated the effect of salt, SPP, and lactate on the growth probabilities and growth or no-growth behavior of E. coli O157:H7 in ground beef and can be used to improve the microbial food safety of ground beef products.     

Validation of time and temperature values as critical limits for the control of Escherichia coli O157:H7 during the production of fresh ground beef

In order to provide beef processors with valuable data to validate critical limits set for temperature during grinding, a study was conducted to determine Escherichia coli o157:H7 growth at various temperatures in raw ground beef. Fresh ground beef samples were inoculated with a cocktail mixture of streptomycin-resistant E. coli O157:H7 to facilitate recovery in the presence of background flora. Samples were held at 4.4, 7.2, and 10 degrees C, and at room temperature (22.2 to 23.3 degrees C) to mimic typical processing and holding temperatures observed in meat processing environments. E. coli O157:H7 counts were determined by direct plating onto tryptic soy agar with streptomycin (1,000 microg/ml), at 2-h intervals over 12 h for samples held at room temperature. Samples held under refrigeration temperatures were sampled at 4, 8, 12, 24, 48, and 72 h. Less than one log of E. coli O157:H7 growth was observed at 48 h for samples held at 10 degrees C. Samples held at 4.4 and 7.2 degrees C showed less than one log of E. coli O157:H7 growth at 72 h. Samples held at room temperature showed no significant increase in E. coli O157:H7 counts for the first 6 h, but increased significantly afterwards. These results illustrate that meat processors can utilize a variety of time and temperature combinations as critical limits in their hazard analysis critical control point plans to minimize E. coli O157:H7 growth during the production and storage of ground beef.     

Use of mustard flour to inactivate Escherichia coli O157:H7 in ground beef under nitrogen flushed packaging

This study was undertaken to determine whether the glucosinolates naturally present in non-deheated mustard flour could serve as a source of allyl and other isothiocyanates in sufficient quantity to kill Escherichia coli O157:H7 inoculated in ground beef at three different levels, during refrigerated storage of the meat under nitrogen. Mustard flour was mixed at 5%, 10% or 20% (w/w) with freshly ground beef, then the beef was inoculated with a cocktail of five strains of E. coli O157:H7 at either 3, 6 or < or =1.6 log10 cfu/g. The ground beef was formed into 100 g patties and each was placed in a bag of Nylon/EVOH/PE, which was back-flushed with 100% N2, heat-sealed and stored at 4 degrees C for < or =21 days. During storage, the allyl isothiocyanate (AIT) levels in package headspaces were determined by gas liquid chromatography. By 21 days, the levels present in treatments were not significantly different. After 21 days storage, there were 0.5, 3 and 5.4 log10 decreases in numbers of E. coli O157:H7 from the initial levels of 6 log10 cfu/g in meat containing 5%, 10% and 20% mustard flour, respectively. When inoculated at 3 log10 cfu/g, E. coli O157:H7 was reduced to undetectable levels after 18, 12 and 3 days with 5%, 10% and 20% mustard flour, respectively. When immunomagnetic separation (IMS) was used for E. coli recovery following its inoculation at < or =1.6 log10 cfu/g, 5% mustard did not completely eliminate the pathogen from ground beef stored for 6 days. The natural microflora of the ground beef which developed in vacuum packages was unaffected by the addition of 5% mustard flour but some inhibition was found at higher concentrations. Sensory evaluation of the cooked ground beef showed that there were no significant differences in the acceptability of meat treated with 5 or 10% mustard flour. However, panelists could distinguish untreated controls from mustard treatments, but considered the mustard-treated meat to be acceptable. These results showed that it is possible to use mustard flour at levels of >5-10% to eliminate E. coli O157:H7 from fresh ground beef.     

Influence of freezing and freezing plus acidic calcium sulfate and lactic acid addition on thermal inactivation of Escherichia coli O157:H7 in ground beef

Undercooked ground beef is a leading vehicle for acquiring Escherichia coli O157:H7 infections through consumption of foods. Studies have been performed to determine the effect of freezing and the combined effect of freezing and addition of a mixture of 20% acidic calcium sulfate (final concentration of 0.4% in ground beef) and 10% lactic acid (final concentration of 0.2% in ground beef) (ACS-LA) on the thermal sensitivity of E. coli O157:H7 in ground beef. Five strains of E. coli O157: H7 were separately inoculated into ground beef and held at 5 degrees C for up to 10 days or -20 degrees C for up to 3 weeks then heated at 57, 60, 62.8, 64.3, and 68.3 degrees C to determine rates of thermal inactivation. Results revealed that D-values (decimal reduction times) at equivalent temperatures for four of five E. coli O157:H7 strains were less in the previously frozen than in the refrigerated ground beef and that strains isolated from ground beef in 1993 and 1994 were generally more sensitive to thermal inactivation than those isolated in 1999 and 2000. Only one strain of E. coli O157:H7 was used to determine the effect of ACS-LA in previously frozen or refrigerated ground beef on rates of thermal inactivation. The addition of ACS-LA to ground beef at 20 ml/kg increased the thermal sensitivity of E. coli O157:H7 in both previously frozen and refrigerated ground beef, with greatest rates of inactivation occurring in previously frozen ground beef containing ACS-LA. D-values at 57 degrees C obtained for E. coli O157:H7 in previously refrigerated and frozen ground beef containing ACS-LA and ACS-LA diluted by half were significantly (P < 0.05) less than those obtained in ground beef with no ACS-LA added. D-values at 60 and 62.8 degrees C were consistently less in ACS-LA treated ground beef, but for most treatments the results were not significantly (P > 0.05) different than the controls. Results revealed that the addition of ACS-LA to ground beef, whether frozen or refrigerated, can reduce the temperature or time required to kill E. coli O157:H7 during heating.     

Distribution of Escherichia coli O157:H7 in beef processed in a table-top bowl cutter

Beef-processing equipment can be contaminated with pathogens such as Escherichia coli O157:H7 and Salmonella spp. The bowl cutter has wide application in particle-size reduction and blending of meat products. This study was undertaken to determine (i) the distribution patterns of E. coli O157:H7 in equipment components and ground beef produced with a table-top bowl cutter under different operational conditions and (ii) the likelihood that pathogen contamination can be transferred to subsequent batches after a batch of beef contaminated with E. coli O157:H7 has been processed in the same bowl cutter. A beef trim (44.6 +/- 29.5 g) inoculated with 2 log CFU of an E. coli O157:H7 mutant strain resistant to rifampicin (E. coli O157:H7rif) was fed by hand into an uncontaminated beef-trim batch under two different batch sizes (2 and 4 kg), three processing times (60, 120, and 240 s), and two feeding modes (running and stoppage fed). There were no significant differences (P > or = 0.05) among all the treatments for the averages of the counts of E. coli O157:H7rif distributed in the ground beef. Regardless of the processing time and the method used to feed the beef trims into the bowl cutter, the whole batch and the following subsequent batch became contaminated when previously contaminated beef was processed. Areas of the bowl cutter most likely to be contaminated with E. coli O157:H7 were (i) the material left on the top of the comb/knife guard and (ii) the knife. Material that overflowed the bowl cutter, when processing the batch with E. coli O157:H7rif, contaminated the equipment surroundings. A Pearson V probability distribution function was determined to describe the distribution of pathogenic organisms in the ground beef, a distribution that can also be applied when conducting process risk analyses on mixing-particle reduction operations for beef trims.     

Evaluating lethality of beef roast cooking treatments against Escherichia coli O157:H7

Added salt, seasonings, and phosphates, along with slow- and/or low-temperature cooking impart desirable characteristics to whole-muscle beef, but might enhance Escherichia coli O157:H7 survival. We investigated the effects of added salt, seasoning, and phosphates on E. coli O157:H7 thermotolerance in ground beef, compared E. coli O157:H7 thermotolerance in seasoned roasts and ground beef, and evaluated ground beef-derived D- and z-values for predicting destruction of E. coli O157:H7 in whole-muscle beef cooking. Inoculated seasoned and unseasoned ground beef was heated at constant temperatures of 54.4, 60.0, and 65.5°C to determine D- and z-values, and E. coli O157:H7 survival was monitored in seasoned ground beef during simulated slow cooking. Inoculated, seasoned whole-muscle beef roasts were slow cooked in a commercial smokehouse, and experimentally determined lethality was compared with predicted process lethality. Adding 5% seasoning significantly decreased E. coli O157:H7 thermotolerance in ground beef at 54.4°C, but not at 60 or 65.5°C. Under nonisothermal conditions, E. coli O157:H7 thermotolerance was greater in seasoned whole-muscle beef than in seasoned ground beef. Meeting U.S. Government (U.S. Department of Agriculture, Food Safety and Inspection Service, 1999, Appendix A) whole-muscle beef cooking guidance, which targets Salmonella destruction, would not ensure ≥6.5-log CFU/g reduction of E. coli O157:H7 in ground beef systems, but generally ensured $ 6.5-log CFU/g reduction of this pathogen in seasoned whole-muscle beef. Calculations based on D- and z-values obtained from isothermal ground beef studies increasingly overestimated destruction of E. coli O157:H7 in commercially cooked whole-muscle beef as process severity increased, with a regression line equation of observed reduction = 0.299 (predicted reduction) + 1.4373.     

Bactericidal effects of Lactobacillus reuteri and allyl isothiocyanate on Escherichia coli O157:H7 in refrigerated ground beef

Two naturally occurring antimicrobial agents were tested in packages of refrigerated ground beef for their ability to reduce the viability of Escherichia coli O157:H7 during storage. Allyl isothiocyanate (AITC) and Lactobacillus reuteri were tested separately and together for their action against a cocktail of five strains of E. coli O157:H7 in ground beef held at 4 degrees C for 25 days. Ground beef prepared from whole, raw inside round beef roasts was inoculated with low (3 log CFU/g) or high (6 log CFU/g) levels of the E. coli O157:H7 mixture. The beef was treated with AITC (about 1,300 ppm), L. reuteri, or both, along with 250 mM of glycerol per kg of meat at two levels (3 and 6 log CFU/g) and according to a design that yielded 8 controls plus 10 different treatments. Samples were analyzed for E. coli O157:H7 survivors, numbers of total bacteria, and lactic acid bacteria on days 0 to 25 at 5-day intervals. L. reuteri at both input levels with glycerol killed E. coli O157:H7 at both inoculated levels before day 20. AITC completely eliminated E. coli O157:H7 at the low-inoculum level (3 log CFU/g) and reduced viability >4.5 log CFU/g at the high-inoculum level (6 log CFU/g) by the end of the storage period. The combination of L. reuteri and AITC did not yield an additive effect against E. coli O157:H7 viability. L. reuteri in the presence of glycerol was highly effective against E. coli O157:H7 in ground beef during refrigerated storage (4 degrees C) in modified atmosphere packages. Sensory testing is planned to evaluate effects of treatments.     

Thermal inactivation of Escherichia coli O157:H7 in ground beef supplemented with sodium lactate

A study was conducted to investigate the antimicrobial effect of sodium lactate (NaL) (0, 1.5, 3.0, and 4.5%) on the survival of Escherichia coli O157:H7 in 93% lean ground beef. Samples inoculated with a mixture of four strains of E. coli O157:H7 (10(7) to 10(8) CFU/g) were subjected to immersion heating in a water bath stabilized at 55, 57.5, 60, 62.5, or 65 degrees C. Results of statistical analysis indicated that the heating temperature was the only factor affecting the decimal reduction times (D-values) of E. coli O157:H7 in 93% lean ground beef. The change in temperature required to change the D-value (the z-value) was determined as 7.6 degrees C. The thermal resistance of this organism was neither affected by the addition of NaL nor by the interactions between NaL and temperature. Adding NaL to ground beef to reduce the thermal resistance of E. coli O157:H7 is therefore not recommended.     

Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR

A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step.     

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.     

Effects of using reduced volumes of nonselective enrichment medium in methods for the detection of Escherichia coli O157:H7 from raw beef

Recent work from our laboratory revealed that tryptic soy broth (TSB) was a superior enrichment medium for use in test-and-hold Escherichia coli O157:H7 methods at levels down to a ratio of three volumes of medium to one volume of sample. Lower ratios were examined for their effect on results obtained from culture isolation, the BAX E. coli O157:H7 MP assay, and the Assurance GDS E. coli O157:H7 assay. Ground beef and boneless beef trim were inoculated with a high level (170 CFU/65 g of ground beef and 43 CFU/65 g of trim) and a low level (17 CFU/65 g of ground beef and 4 CFU/65 g of trim) of E. coli O157:H7 and enriched in 3, 1, 0.5, and 0 volumes of TSB. The volume of TSB used did not affect E. coli O157:H7 detection by culture isolation, Assurance GDS detection in ground beef or trim, or the BAX MP assay detection in ground beef. However, BAX MP assay detection of E. coli O157:H7 in beef trim was 50, 42, and 33% positive when enrichment volumes of 0.5x, 1x, and 3x, respectively, were used. Optimum results with all methods were obtained using 1 volume of TSB. We concluded that detection test results can be considered valid as long as enrichment medium is used, even when it is less than the specified 3 or 10 volumes.     

Effect of food matrix and cell growth on PCR-based detection of Escherichia coli O157:H7 in ground beef

The purpose of this work was (i) to investigate the feasibility of a previously reported upstream processing method for PCR template preparation to facilitate the detection of Escherichia coli O157:H7 from ground beef and (ii) to assess the impact of cell growth (no growth in the matrix versus growth in the matrix) on molecular detection limits. Two food matrices (autoclaved and raw ground beef) were evaluated in all studies. For no-growth experiments, 10-g meat samples were inoculated with 10(2) to 10(7) CFU/g E. coli O157:H7 and then homogenized. The homogenates were processed to remove large particulates and inhibitors using a two-phase upstream processing method consisting of two sequential centrifugation steps, the second of which used titanous hydroxide to facilitate bacterial immobilization. After upstream processing, sample concentrates were extracted for DNA isolation and amplified by PCR. For growth experiments, 10-g meat samples were inoculated at 1 CFU of E. coli O157:H7 per gram, allowed to grow to 10(2) to 10(7) CFU/g, and then processed for PCR assay. Cell recoveries after upstream processing ranged from 15.9 to 77.6% and were not facilitated by the use of titanous hydroxide, as compared with a saline control (P > 0.05). Bacterial cell recovery and PCR detection limits were similar when comparing autoclaved ground beef and raw ground beef, but cell recoveries were highly variable for raw ground beef samples in which E. coli O157:H7 cells were allowed to grow before processing for detection. Overall, PCR detection limits approximated 10(3) CFU/g of ground beef for all treatments. These results indicate that use of model food systems may not always provide an accurate replication of real-world conditions when evaluating PCR detection limits.     

Inactivation of Escherichia coli O157:H7 in packaged ground beef by allyl isothiocyanate

Commercial allyl isothiocyanate (AIT) was examined for its ability to reduce numbers of Escherichia coli O157:H7 inoculated in fresh ground beef packaged under nitrogen and stored refrigerated or frozen. A five-strain cocktail of E. coli O157:H7 containing 3 or 6 log10 cfu/g was inoculated into 100 g ground beef and formed into 10x1-cm patties. A 10-cm diameter filter paper disk treated with AIT suspended in sterile corn oil was placed on top of a single patty. One patty and paper disk were placed in a bag of Nylon/EVOH/PE with O2 permeability of 2.3 cm3 m(-2) 24 h atm at 23 degrees C. The bags were back-flushed with 100% nitrogen, heat-sealed and stored at 10, 4 and -18 degrees C for 8, 21 or 35 days, respectively. During storage, the AIT levels in the package headspaces were determined by gas liquid chromatography, and mesophilic bacteria and E. coli O157:H7 were counted. The mesophilic aerobic bacteria in ground beef patties were largely unaffected by the addition of AIT. At an initial population of 3 log10 cfu/g, E. coli O157:H7 was reduced by AIT to undetectable levels after 18 days at 4 degrees C or 10 days at -18 degrees C. In samples inoculated with 6 log10 cfu/g, a >3 log10 reduction of E. coli O157:H7 was observed after 21 days at 4 degrees C, while a 1 log10 reduction was observed after 8 and 35 days at 10 and -18 degrees C, respectively. The final AIT concentrations in the headspaces after storage at 10, 4, and -18 degrees C were 444, 456, and 112 microg/ml at 8, 21, and 35 days, respectively. Results showed that AIT can substantially reduce numbers of E. coli O157:H7 in fresh ground beef during refrigerated or frozen storage.     

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.     

Rapid detection of Escherichia coli O157:H7 inoculated in ground beef,chicken carcass,and lettuce samples with an immunomagnetic chemiluminescence fiber-optic biosensor

A biosensor was evaluated with regard to its usefulness in the rapid detection of Escherichia coli O157:H7 inoculated in ground beef, chicken carcass, and romaine lettuce samples. The biosensor consisted of a chemiluminescence reaction cell, a fiber-optic light guide, and a luminometer linked to a personal computer in conjunction with immunomagnetic separation. The samples inoculated with E. coli O157:H7 were first centrifuged and suspended in buffered peptone water and then incubated with anti-E. coli O157 antibody-coated magnetic beads and horseradish peroxidase (HRP)-labeled anti-E. coli O157 antibodies to form antibody-coated bead-bacterium-HRP-labeled antibody sandwich complexes. Finally, the sandwich complexes were separated from the samples in a magnetic field and reacted with luminol in the reaction cell. The number of E. coli O157:H7 cells was determined by collecting the HRP-catalyzed chemiluminescence signal from the bead surface through a fiber-optic light guide and measuring the signal with a luminometer. The chemiluminescence biosensor was specific for E. coli O157:H7 in samples containing other bacteria, including Salmonella Typhimurium, Campylobacter jejuni, and Listeria monocytogenes. The chemiluminescence signal was linear on a log scale from 10(2) to 10(5) CFU of E. coli O157:H7 per ml in samples. Detection could be completed within 1.5 h without any enrichment. The detection limits for ground beef, chicken carcass, and lettuce samples were 3.2 x 10(2), 4.4 x 10(2), and 5.5 x 10(2) CFU of E. coli O157:H7 per ml, respectively.     

Empirical Distribution Models for Escherichia coli 57:H7 in Ground Beef Produced by a Mid-size Commercial Grinder

ABSTRACT: The purpose of this research was to develop empirical models that describe the amount and distribution of ground beef contaminated with Escherichia coli O157:H7 when a contaminated beef trim is introduced into a batch of uncontaminated beef before processing in a mid-size commercial grinder (34 g/s). A beef trim was inoculated with a rifampacin-resistant strain of E. coli O157:H7 and added to a batch of noncontaminated trims at the grinding step. To study the distribution of the E. coli O157:H7^rif in the ground beef, 6 treatments with different inoculum levels (1 to 6 log₁₀ colony-forming units [CFU]) were tested. Removal or pick up of the residual contamination with E. coli O157:H7^rif left in the grinder was evaluated. E. coli O157:H7^rif was detected in 9% to 86% of the total ground beef for the 1 to 6 log₁₀ CFU inoculum levels, respectively. E. coli O157:H7^rif contamination was detected in the collar that fixes the grinder's die and blade to the hub. An exponential algorithm described the relationship between the quantities of ground beef containing E. coli O157:H7^rif and the inoculum level ( R ²= 0.82). Distribution models based on a Chi-squared algorithm were developed for each inoculum level describing the contamination level as a function of the batch fraction processed ( R ²= 0.81 to 0.99). The results of this study corroborate that when beef processors test for pathogenic contamination in a mid-scale grinder, they should test the beef residues in the collar that fixes the grinder's die and blade to the hub.